Metabolites Profiling and In Vitro Biological Characterization of Different Fractions of Cliona sp. Marine Sponge from the Red Sea Egypt

Red Sea marine sponges are an important source of biologically active natural products. Therefore, the present study aimed to investigate, for the first time, the components of n-hexane, dichloromethane, and ethyl acetate fractions of Cliona sp. marine sponge collected from the Red Sea, Egypt using UPLC-ESI-MS/MS (Ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry) analysis. The analysis revealed the tentative identification of 23, 16, and 24 compounds from the n-hexane, dichloromethane, and ethyl acetate fractions of Cliona sp., respectively. In addition, the examination of these fractions resulted in the isolation and identification of three sterols and one amino acid. The identification of the isolated compounds was confirmed by 1D and 2D NMR (Nuclear Magnetic Resonance), and MS (Mass spectrometry), and IR (Infrared) spectroscopy. The in vitro cytotoxic, antioxidant, and antimicrobial activities of the total ethanolic extract and its sub-fractions were also evaluated. Interestingly, the ethyl acetate fraction showed potent cytotoxic activity against colon (HCT-116) and human larynx carcinoma (HEP-2) cell lines with IC50 (Half-maximal Inhibitory Concentration) 6.11 ± 0.2 and 12.6 ± 0.9 µg/mL, respectively. However, the dichloromethane fraction showed strong antioxidant activity, with IC50 75.53 ± 3.41 µg/mL. Notably, the total ethanolic extract showed the strongest antibacterial activity against Staphylococcus aureus and Escherichia coli, with MIC (Minimum Inhibitory Concentration) 62.5 ± 0.82 and 125 ± 0.62 µg/mL, respectively, compared to other fractions. In conclusion, this is the first report on the secondary metabolites content and biological activities of Cliona sp. from the Red Sea, Egypt. It also highlights the need for further research on the most active fractions against various cancer cell lines and resistant bacterial and fungal strains. Cliona sp. extract and its fractions could be a potential source of novel and safe natural drugs with a wide range of medicinal and pharmaceutical applications.


Introduction
The Red Sea is a valuable and variant biosphere. Around 2000 km of coral reef runs along the Red Sea's coastline, which is inhabited by various invertebrate species. The huge biodiversity and limited research make the Red Sea an unexplored goldmine for the innovation of novel biologically active marine drugs. Fifty-eight percent of the reported marine organisms are collected from Egypt due to the great biodiversity of the Egyptian environment. Notably, marine sponges are the most frequently collected organisms either (1), brassicasterol (2) [20][21][22], stigmasterol (3) [23], and taurine (4) [24,25] (Figure 1). To the best of our knowledge, this is the first report about the isolation of these compounds from Cliona sp. Compounds 1 and 2 were obtained as a white amorphous mixture and were identified as coprostanol and brassicasterol based on a comparison of their spectroscopic analysis data, including EI-MS ( Figure S1), GC-MS ( Figures S2-S4), the ratio of coprostanol to brassicasterol (88.5% to 11.5%), 1 H-NMR, and 13 C-NMR (Table S1), with the reported literature data [20][21][22].
Compound 3 was isolated as a crystalline white powder with a melting point of 162 °C and was freely soluble in dichloromethane. It was identified as stigmasterol based on a comparison of its EI-MS ( Figure S5), 1 H-NMR, and 13 C-NMR (Table S2) with the reported data [23].
Compound 4 is colorless needle crystals with a melting point of 300 °C and is soluble in hot methanol. It was identified as taurine based on a comparison of its EI-MS ( Figure  S6), 1 H-NMR, and 13 C-NMR with the reported literature [24,25].

Tentative Identification of Constituents of Cliona sp. Fractions by UPLC-ESI-MS/MS (Ultra-Performance Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry)
In the current study, UPLC-ESI-MS/MS in positive ion mode was used to analyze Cliona sp. From the Red Sea, Egypt n-hexane (CH), dichloromethane (CD), and ethyl acetate (CE) fractions. The compounds' identification was based on their MS 2 data given by the precursor ion mass, their fragments, and their neutral mass loss, as well as comparison with the available literature. Additionally, investigation of unidentified peaks from the total extract and fractions is currently underway.  Compounds 1 and 2 were obtained as a white amorphous mixture and were identified as coprostanol and brassicasterol based on a comparison of their spectroscopic analysis data, including EI-MS ( Figure S1), GC-MS ( Figures S2-S4), the ratio of coprostanol to brassicasterol (88.5% to 11.5%), 1 H-NMR, and 13 C-NMR (Table S1), with the reported literature data [20][21][22].
Compound 3 was isolated as a crystalline white powder with a melting point of 162 • C and was freely soluble in dichloromethane. It was identified as stigmasterol based on a comparison of its EI-MS ( Figure S5), 1 H-NMR, and 13 C-NMR (Table S2) with the reported data [23].
Compound 4 is colorless needle crystals with a melting point of 300 • C and is soluble in hot methanol. It was identified as taurine based on a comparison of its EI-MS ( Figure S6), 1 H-NMR, and 13 C-NMR with the reported literature [24,25].
Compounds 1, 2, and 3 (Rt. 0.48, 0.66, and 0.67 min, respectively) generated a protonated molecular ion peak at m/z 133 [M + H] + . They also gave a diagnostic MS 2 fragment ion at m/z 88 [M + H-COOH] + corresponding to the loss of the carboxylic group, in addition to the fragment ion at m/z 73 for C2H4NO2, showing the subsequent loss of COOH, NH2 moieties. According to the literature, compounds 1, 2, and 3 are tentatively identified as asparagine and its two isomers [26].  Figure 4B). Furthermore, fragment ion at m/z 43 [59-NH2] + corresponds to the loss of the amino group. With these data, with the aid of the literature, compounds 4 and 5 were tentatively identified as L-serine with its isomer [26].
Compound 6 (Rt. 18.18 min) showed ESI-MS 1 at m/z 301 [M + Na] + and m/z 279 [M + H] + , and fragment ion at m/z 149, which showed a loss of 130 amu corresponding to C4H9 and C4H9O moieties. Accordingly, this compound was tentatively identified as dibutyl phthalate [27,28].  The MS 2 showed a daughter ion at m/z 184, corresponding to protonated phosphocholine moiety. The base peak fragment at m/z 104 [184-HPO3] + showed a loss of phosphate moiety from the phosphocholine base. The fragment ion at m/z 124 was assigned for the loss of N(CH3)3 from phosphocholine moiety, which was confirmed by the presence of the fragment ion at m/z 60. Unfortunately, ions reflecting the fatty acid constituent and the identities of the sphingosine base were not observed as it was cleaved together, leaving phosphocholine moiety;   3 ] + showed a loss of phosphate moiety from the phosphocholine base. The fragment ion at m/z 124 was assigned for the loss of N(CH 3 ) 3 from phosphocholine moiety, which was confirmed by the presence of the fragment ion at m/z 60. Unfortunately, ions reflecting the fatty acid constituent and the identities of the sphingosine base were not observed as it was cleaved together, leaving phosphocholine moiety; therefore, compound 7 was tentatively identified as a sphingomyelin derivative [29].
Compound 8 (R t . 27.74 min) was tentatively identified as aflatoxin G. It generated ESI-MS 1 at m/z 353 [M + Na] + and MS 2 fragment ion at m/z 295 [M + H-OCH 3 -CO] + , corresponding to the loss of a methoxy and carbonyl groups. Additionally, a daughter ion at m/z 275 showed the loss of two carbonyl groups beside sodium. Furthermore, the fragment ion at m/z 243 showed the loss of two oxygen atoms from 275 amu, while the fragment ion at m/z 228 showed the subsequent loss of the CH 3 group. Through comparison with the available literature, compound 8 was tentatively identified as aflatoxin G2 [30][31][32].
Compound 10 (R t . 28.08 min) showed an ESI-MS protonated molecular ion peak at m/z 281 [M + H] + . The cleavage of the seven-membered ring gave two important fragment ions at m/z 120 and 161. Both fragments subsequently gave a fragment ion at m/z 104 amu after the loss of NH from the fragment ion at m/z 120 amu and the loss of CO and NCH 3 from the other fragment at m/z 161. Additionally, another fragment ion was observed at m/z 223 [M + H-CONCH 3 ] + which, by the loss of benzoyl moiety, gave a characteristic fragment ion at m/z 133 amu. The mass data of this compound are in good agreement with the reported literature on cyclopeptin [33].

Characterization of the Components of Cliona sp. Dichloromethane Fraction
The HPLC-ESI-MS/MS analysis of the methylene chloride fraction of the Red Sea sponge Cliona sp. led to the tentative identification of 15 compounds, as shown in Figure 5, Figure 6 and Table 2.

Characterization of the Components of Cliona sp. Dichloromethane Fraction
The HPLC-ESI-MS/MS analysis of the methylene chloride fraction of the Red Sea sponge Cliona sp. led to the tentative identification of 15 compounds, as shown in Figure  5, Figure 6 and Table 2.     was also detected. Therefore, compound 5 was tentatively identified as N-methyl leucine [26].
Compounds 6 and 7 (Rt. 0.55 and 0.67 min, respectively) generated a protonated molecular ion peak at m/z 133 [M + H] + . They also gave diagnostic MS 2 fragment ions at m/z 88 [M + H-COOH] + , which corresponded to the loss of the carboxylic group. In addition, the fragment ion at m/z 73 for C 2 H 4 NO 2 showed the subsequent loss of C 2 H 4 NO moiety. According to the literature [41], compounds 6 and 7 were tentatively identified as asparagine and its isomer [26].
Compound 9 (R t . 25.16 min.) exhibited a protonated molecular ion peak at m/z 353 [M + H] + . MS 2 ions were observed at m/z 321 and 257, corresponding to the loss of the methoxy group and chloride atom, respectively. A fragment at m/z 215 was observed, corresponding to C 8 H 7 O 4 Cl, while the fragments at m/z 165 are due to C 7 H 4 O 4 . Therefore, compound 9 was tentatively identified as griseofulvin [42].  Figure 4D). With these data, and with the aid of the available literature, compound 10 was tentatively identified as β-sitosterol [43]. of all of these compounds showed daughter ions at m/z 184, corresponding to protonated phosphocholine moiety. The base peak fragment at m/z 104 [184-HPO 3 ] + showed a loss of phosphate moiety from the phosphocholine base. The fragment ion at m/z 124 was assigned for the loss of the N (CH 3 ) 3 moiety, which was confirmed by the presence of a fragment ion at m/z 60. Unfortunately, fragment ions reflecting the fatty acid constituent and the identities of the sphingosine base were not observed, as they were cleaved together, leaving phosphocholine moiety; therefore, the four compounds were tentatively identified as sphingomyelin derivatives [29].
Compound 15 (R t 5.05 min) was tentatively identified as an isoleucine derivative from ESI-MS and MS 2 spectral data. The molecular ion peak was formed at m/z 216 and the MS 2 spectrum showed fragment ions at m/z 132 [M + H-R] + , corresponding to the loss of R-moiety and give amu of isoleucine. The prominent base peak produced at m/z 86 [M + H-R-COOH] + showed a loss of the carboxyl group, confirming the presence of isoleucine. According to these data, compound 15 was tentatively identified as an isoleucine derivative. [26].
Compound 17 (R t 6.40) generated its ESI-MS molecular ion peak at m/z 198 [M + H] + . The MS 1 fragment ion produced at m/z 144 showed the loss of three molecules of water in addition to fragment ions at m/z 111 due to the loss of the side chain C 3 H 6 O 2 N. From these data, and through comparison with the available literature, compound 17 was tentatively identified as L-3-(3,4-dihydroxyphenyl)-alanine (Dopa) [26].  are the active fractions against P. aeruginosa with MIC 125 ± 0.92 and 500 ± 0.32 µg/mL, respectively. Interestingly, the CD fraction is the only fraction that revealed strong antifungal activity against C. albicans with MIC 500 ± 0.9 µg/mL compared to the fluconazole (positive control) with MIC values 50 ± 0.24 µg/mL. The significant antimicrobial and cytotoxic activities might be attributed to the presence of different phytosterols (β-sitosterol, stigmasterol, coprostanol, and brassicasterol) and sphingomyelin derivatives with reported potent antibacterial activity [54,55].  The antimicrobial activity of the extract based on the MIC values was classified as follows: 50-500 µg/mL = strong activity; 600-1500 µg/mL = moderate activity; and >1500 µg/mL = weak activity or inactive [53]. According to the previous classification of Cliona sp., the total alcoholic extract (CT), dichloromethane (CD), and ethyl acetate (CE) fractions showed strong antibacterial activity against S. aureus only with MIC values of 62.5 ± 0.82, 125 ± 0.58, and 250 ± 0.88 µg/mL, respectively, compared to ciprofloxacin (positive control) with MIC values of 1.56 ± 1.2 µg/mL for Gram-positive bacteria and 3.125 ± 0.89 µg/mL for Gram-negative bacteria. Additionally, CT, CD, and CE are active against E. coli, with MIC 125 ± 0.62, 125 ± 0.72, and 250 ± 0.98 µg/mL, respectively. Notably, only CD and CE are the active fractions against P. aeruginosa with MIC 125 ± 0.92 and 500 ± 0.32 µg/mL, respectively. Interestingly, the CD fraction is the only fraction that revealed strong antifungal activity against C. albicans with MIC 500 ± 0.9 µg/mL compared to the fluconazole (positive control) with MIC values 50 ± 0.24 µg/mL. The significant antimicrobial and cytotoxic activities might be attributed to the presence of different phytosterols (β-sitosterol, stigmasterol, coprostanol, and brassicasterol) and sphingomyelin derivatives with reported potent antibacterial activity [54,55]. Table 4. Antimicrobial activity of Cliona sp. Total extract (CT), Hexane (CH), Dichloromethane (CD), and Ethyl acetate (CE) fractions by agar diffusion method.

Antioxidant Activity
Sponges and seaweeds represent an abundant source of natural antioxidants [56]. In the present work, the antioxidant activity of the Cliona sp. total extract (CT), n-hexane (CH), dichloromethane (CD), and ethyl acetate (CE) fractions are evaluated using the DPPH method and ascorbic acid as a positive control ( Figure 10). DPPH in methanol (without the tested sample) was used as a negative control. Data were analysed by using one-way ANOVA and statistical significance was calculated with Dunnett's multiple comparisons test, and the significance level compared to the control is indicated by asterisks (*, p < 0.05; ***, p < 0.0001). The data display the mean ±SD of three biological replicas.

Cytotoxic Activity
In the current study, an MTT assay was used to assess the cytotoxic activity of Cliona sp. total extract (CT), n-hexane (CH), dichloromethane (CD), and ethyl acetate (CE) fractions against HCT-116 (Colon carcinoma) and HEP-2 (larynx carcinoma) cell lines using a concentration range of 0-500 µg/mL. The potency of the cytotoxic substances based on the IC50 values was classified as follows: IC50 ≤ 20 µg/mL is highly active, IC50 21-200 µg/mL is moderately active, IC50 201-500 µg/mL is weakly active, and IC50 > 501 µg/mL is inactive, which is consistent with the American National Cancer Institute protocol [58]. The tested extract (CT) and fractions (CH, CD, and CE) ( Figure 11 and Table 5) showed remarkable cytotoxic activity against the investigated cell lines with an IC50 range from 6.11 ± 0.2 to 69.7 ± 3.41 µg/mL for HCT-116 (colon carcinoma) and from 12.6 ± 0.9 to 116.5 ± 4.09 µg/mL DPPH in methanol (without the tested sample) was used as a negative control. Data were analysed by using one-way ANOVA and statistical significance was calculated with Dunnett's multiple comparisons test, and the significance level compared to the control is indicated by asterisks (*, p < 0.05; ***, p < 0.0001). The data display the mean ±SD of three biological replicas.
The tested extract and its fractions showed a concentration-dependent antioxidant activity by an increase in their DPPH radical scavenging percentage as demonstrated in Figure 10A. The IC 50 values (the concentration required to scavenge DPPH by 50%) are presented in Figure 10B. Notably, the smaller the IC 50 , the higher the scavenging activity, and it was reported by [46] that the tested extract or fraction is considered a weak antioxidant when the IC 50 values are in the range of (151-200), moderate when the values are in the range (100-150), strong when the values are in the range (50-100), and very strong when the IC 50 values are <50. According to the previous classification, the dichloromethane fraction showed strong DPPH scavenging activity with IC 50 75.53 ± 3.41 µg/mL, followed by the total extract (CT) and the n-hexane fraction (CH) with IC 50 149.2 ± 4.85 and 198.84.2 ± 8.23 µg/mL, respectively.
However, the ethyl acetate fraction of the Cliona sp. has a very weak antioxidant potential, with IC 50 1025.13 ± 32.79 µg/mL, compared to ascorbic acid as a positive control with IC 50 10.6 ± 0.8 µg/mL. The activity of the dichloromethane fraction is probably due to the presence of β sitosterol and stigmasterol.
Marine sterols' antioxidant activity has been demonstrated by their ability to normalize various oxidative markers and to promote the expression of enzymatic and non-enzymatic antioxidants. Furthermore, they have structure and function similarities with biological sterols, especially cholesterol. Stigmasterol and β-sitosterol, specifically, have shown very promising results in clinical trials against several diseases [57]. Marine sterols may also provide prospective lead compounds in the development of new therapeutic drugs, due to recent advances in technology such as nanoparticles and microencapsulation [57].

Cytotoxic Activity
In the current study, an MTT assay was used to assess the cytotoxic activity of Cliona sp. total extract (CT), n-hexane (CH), dichloromethane (CD), and ethyl acetate (CE) fractions against HCT-116 (Colon carcinoma) and HEP-2 (larynx carcinoma) cell lines using a concentration range of 0-500 µg/mL. The potency of the cytotoxic substances based on the IC 50 values was classified as follows: IC 50 ≤ 20 µg/mL is highly active, IC 50 21-200 µg/mL is moderately active, IC 50 201-500 µg/mL is weakly active, and IC 50 > 501 µg/mL is inactive, which is consistent with the American National Cancer Institute protocol [58]. The tested extract (CT) and fractions (CH, CD, and CE) ( Figure 11 and Table 5) showed remarkable cytotoxic activity against the investigated cell lines with an IC 50 range from 6.11 ± 0.2 to 69.7 ± 3.41 µg/mL for HCT-116 (colon carcinoma) and from 12.6 ± 0.9 to 116.5 ± 4.09 µg/mL for HEP-2 (larynx carcinoma). Interestingly, the Cliona sp. ethyl acetate fraction (CE) showed potent cytotoxic activity with IC 50 6.11 ± 0.2 and 12.6 ± 0.9 µg/mL for HCT-116 and HEP-2 cell lines, respectively, compared to vinblastine (positive control) with IC 50 2.34 ± 0.28 and 6.61 ± 0.59 for HCT-116 and HEP-2, respectively. The total extract, as well as the other fractions, exhibited moderate cytotoxic activity in the following order: CD > CH > CT [58].
According to the UPLC-ESI-MS/MS analysis, the ethyl acetate fraction (CE) revealed the presence of several compounds with promising reported anticancer activity, particularly stachydrine (proline betain) and taurine (2-aminoethanesulfonic acid). Several research studies have demonstrated the potent anticancer properties of stachydrine in a wide range of cancer types, including colon cancer [59], breast cancer [60], gastric cancer [61], and prostate cancer [62], by preventing cell migration and invasion, inhibiting cell proliferation, and triggering apoptosis [63]. In addition, various reports have documented the antioxidant, hypoglycemic, and anti-inflammatory properties of taurine. Taurine not only reduces the chemotherapy's side effects but also causes cytotoxic activity by preventing cell proliferation and triggering apoptosis [64].
Consistent with our results, several extracts or fractions of sponges collected from the Red Sea have cytotoxic activities such as Xestospongia testudinaria [65], Haliclona sp. [66], Spheciospongia vagabunda, [67], and Hyrtios erectus [68]. According to the UPLC-ESI-MS/MS analysis, the ethyl acetate fraction (CE) revealed the presence of several compounds with promising reported anticancer activity, particularly stachydrine (proline betain) and taurine (2-aminoethanesulfonic acid). Several research studies have demonstrated the potent anticancer properties of stachydrine in a wide range of cancer types, including colon cancer [59], breast cancer [60], gastric cancer [61], and prostate cancer [62], by preventing cell migration and invasion, inhibiting cell proliferation, and triggering apoptosis [63]. In addition, various reports have documented the antioxidant, hypoglycemic, and anti-inflammatory properties of taurine. Taurine not only reduces the chemotherapy's side effects but also causes cytotoxic activity by preventing cell proliferation and triggering apoptosis [64].

General Materials and Methods
A UV lamp was used for thin layer chromatography (TLC) visualization: UVP, GL-58 (λ max 254 and 366 nm). A circulating hot-air oven, WT-binder 7200 (Tuttlingen, Germany), was used in this study.
Nuclear magnetic resonance (NMR) experiments, 1D and 2D analyses, were carried out using a Bruker AMX 400 MHz (Billerica, MA, USA) for 1 H NMR and with standard pulse sequences operating at 100 MHz for 13 C-NMR. 1 H-13 C one-bond connectivity was detected with the HSQC gradient pulse factor selection. Two-and three-bond connectivities were identified by the HMBC experiment. Coupling constants (J) are reported in Hz and chemical shifts are reported in δ (ppm) unless otherwise mentioned. The internal standard is Tetramethylsilane. The spectroscopic grade of DMSO-d 6 (solvent at room temperature) was used for spectral analysis.

Collection of Marine Sponge Samples
Marine sponges, namely Cliona sp. Class Demospongiae, were collected from the Red Sea, 20 km away from Sharm El sheikh [27 • 45 57.8 N 34 • 22 10.8 E] by scuba diving at a depth of 8:10 m off, during November-December 2018 ( Figure 12). The collected material was immediately frozen and kept at −20 • C until investigation. Saad Zakaria, Marine Science Department, Faculty of Science, Suez Canal university defined the sponges' biomass.
pulse sequences operating at 100 MHz for 13 C-NMR. 1 H-13 C one-bond connectivity was detected with the HSQC gradient pulse factor selection. Two-and three-bond connectivities were identified by the HMBC experiment. Coupling constants (J) are reported in Hz and chemical shifts are reported in δ (ppm) unless otherwise mentioned. The internal standard is Tetramethylsilane. The spectroscopic grade of DMSO-d6 (solvent at room temperature) was used for spectral analysis.

Collection of Marine Sponge Samples
Marine sponges, namely Cliona sp. Class Demospongiae, were collected from the Red Sea, 20 km away from Sharm El sheikh [27°45′57.8″N 34°22′10.8″E] by scuba diving at a depth of 8:10 m off, during November-December 2018 ( Figure 12). The collected material was immediately frozen and kept at −20 °C until investigation. Saad Zakaria, Marine Science Department, Faculty of Science, Suez Canal university defined the sponges' biomass.

Extraction and Fractionation of Cliona sp.
The fresh sponge material Cliona sp. (2840 g wet weight) was frozen immediately after collection. The sponge material was chopped while frozen into small pieces and shed dried for 48 h. It was then extracted with absolute ethanol (3 × 3 L) at room temperature. The combined crude extract was evaporated under vacuum, and the concentrated extract (91.2 g) was dispersed in water/methanol (9:1) and partitioned successively with n-hexane, dichloromethane, and ethyl acetate to obtain n-hexane, (16.67 g), dichloromethane, (13 g) and ethyl acetate (0.7 g) fractions.

Extraction and Fractionation of Cliona sp.
The fresh sponge material Cliona sp. (2840 g wet weight) was frozen immediately after collection. The sponge material was chopped while frozen into small pieces and shed dried for 48 h. It was then extracted with absolute ethanol (3 × 3 L) at room temperature. The combined crude extract was evaporated under vacuum, and the concentrated extract (91.2 g) was dispersed in water/methanol (9:1) and partitioned successively with n-hexane, dichloromethane, and ethyl acetate to obtain n-hexane, (16.67 g), dichloromethane, (13 g) and ethyl acetate (0.7 g) fractions. The elution of the column was carried out with n-hexane and the polarity was gradually increased using dichloromethane followed by methanol. Fractions eluted with 70% n-hexane /CH 2 Cl 2 were evaporated to afford (3.6 g) residue, which was chromatographed on the silica gel column (50 g, 40 × 2 cm) for purification. The elution was started with n-hexane followed by ethyl acetate, and then methanol in a gradient elution manner. Important fractions eluted with 95% n-hexane in ethyl acetate were concentrated under a vacuum and subjected to crystallization from hot methanol to afford a mixture of compounds 1 and 2 (93 mg). The mixture was obtained as a white amorphous powder that is freely soluble in dichloromethane.

Isolation of Compound 3 from Dichloromethane Soluble Fraction of Cliona sp.
About 11 g of dichloromethane soluble fraction of Cliona sp. was placed on the top of the silica gel column. The elution was started with n-hexane and the polarity was gradually increased using ethyl acetate followed by methanol. The collected fractions were concentrated under reduced pressure. Compound 3, (7 mg) crystalline white powder, freely soluble in dichloromethane, was isolated from the fractions eluted with 97% n-hexane /CH 2 Cl 2 .

Isolation of Compound 4 from Ethyl Acetate Soluble Fraction of Cliona sp.
About 0.5 g of ethyl acetate soluble fraction of Cliona sp. was placed on the top of sephadex (LH-20) column and was eluted with n-hexane and methanol. Twenty-six fractions (10 mL, each) were collected. Combined fractions (11)(12) were concentrated and crystallized using hot methanol/acetone to give 3 mg of colorless needle-shaped crystals soluble in hot methanol designated as compound 4.

UPLC-ESI-MS/MS Analysis and Separation Method of Cliona sp. Extract and Its Fractions
Cliona sp. extract, and CH, CD, and CE fractions, were analyzed in an ACQUITY UPLC-BEH coupled to a XEVO TQD triple quadruple Mass Spectrometer (Waters Corporation ® , Milford, MA, USA), which was equipped with an electrospray ionization (ESI) source functioning with positive polarity at a mass range of m/z 100-1000 atomic mass units. The tested extract and fractions were prepared at a concentration of 0.2-0.5 mg/mL in HPLC methanol and filtered through a 0.2 µm membrane disc filter, and then 10 µL of the sample was injected. The constituents were separated using a binary LC solvent system controlled by MassLynx software (version 4.1) Waters Corporation (Milford, MA, USA) to analyze the MS and MS 2 data. The reversed-phase separations were performed by an ACQUITY UPLC-BEH column [silica C 18 , 1.7 µm (particle size), 2.1 × 50 mm (inner diameter), Waters ® ] eluted with H 2 O + 0.1% formic acid (A) and Methanol+ 0.1% formic acid (B), HPLC grade, at 0.2 mL/min flow rate with the same gradient used by [19,69] as follows: 0-2 min 10% B isocratic; 2-5 min, linear gradient B 10 to 30%; 5-15 min, linear gradient from 30% to 70% B; 15-22 min, linear gradient from 70% to 90% B; 22-25 min, 90% B isocratic. Finally, the process included washing and reconditioning the column. The ESI parameters in positive ionization mode were adjusted as follows: source temperature 150 • C; cone voltage 30 eV; capillary voltage 3 kV; desolvation temperature 440 • C; cone gas flow 50 L/h; and desolvation gas flow 900 L/h. The MS 2 settings were kept at 30 eV of collision energy.

GC-MS Analysis of Compounds 1 & 2
Compounds 1 and 2 were subjected to GC/MS analysis according to [70] with minor modification. GC/MS Analysis Mass spectra were recorded using (Shimadzu GCMS-QP2010, Kyoto, Japan) equipped with Rtx-5MS ® fused bonded column (30 m × 0.25 mm i.d.,× 0.25 µm film thickness) (Restek Inc., Edmond, OK, USA) equipped with a split-splitless injector. The initial column temperature was kept at 50 • C for 3 min and programmed to 300 • C at a rate of 5 • C/min and kept constant at 300 • C for 10 min. Injector temperature was 280 • C. Helium carrier gas flow rate was 1.37 mL/min. All the mass spectra were recorded applying the following condition: (equipment current) filament emission current, 60 mA; ion source, 220 • C. Ionization voltage, 70 eV; diluted samples (1% v/v) were injected with split mode (1:15, split ratio).

Biological Activities
The antioxidant, cytotoxic, and antimicrobial activities of the Clion sp. extract and CH, CD, and CE fractions were done at the Regional Center for Mycology and Biotechnology (RCMB) at the Al-Azhar University.

Antioxidant Activity
The antioxidant activity of the CT extract, and CH, CD, and CE fractions, was determined by the DPPH free radical scavenging assay as described by [19,71,72] with minor modifications. Briefly, DPPH (2,2-diphenyl-1-picrylhydrazyl) radical in methanol (0.004% w/v) was freshly prepared and stored at 10 • C in the dark. Different concentrations (2.5-1280 µg/mL) of the tested extract and its fractions in methanol were also prepared. Forty microliters of aliquot of the methanol solution was added to 3 mL of DPPH solution and allowed to stand for 10 min at room temperature in the dark. Absorbance measurements were recorded immediately with a UV-visible spectrophotometer (Milton Roy, Spectronic 1201, Houston, TX, USA). The decrease in absorbance at λ max 515 nm was measured continuously, with data being recorded at 1 min intervals until the absorbance stabilized (16 min). The absorbance of the DPPH radical without antioxidant (negative control) and the reference compound ascorbic acid (positive control) were also measured. All the determinations were performed in three replicates and averaged. The percentage inhibition (PI) of the DPPH radical was calculated according to the equation: where AC = absorbance of the control at t = 0 min and AT = absorbance of the sample + DPPH at t = 16 min [73]. The DPPH radical scavenging percentage was plotted against each sample concentration and ascorbic acid (µg/mL) to measure the antioxidant capacity (IC 50 ), which resulted in a 50% reduction in the DPPH solution absorbance from its initial absorbance. The lower the IC 50 , the stronger antioxidant activity.

Cytotoxic Activity
Cliona sp. extract, and CH, CD, and CE fractions, were evaluated for their cytotoxic activity in two cell lines against HEB-2 and HCT-116 (Human larynx carcinoma and Colon carcinoma) using MTT assay as explained by [19,74,75]. Vinblastine sulphate and DMSO (Dimethyl sulfoxide) were used as positive and negative controls, respectively. The tested cell lines were obtained from VACSERA Tissue Culture Unit Giza, Egypt. DMEM (Dulbecco's Modified Eagle's Medium) was used for the tested cells' propagation. Stock solutions of the extract and fractions were prepared in 10% DMSO in ddH 2 O. The cytotoxicity was determined using the MTT assay as described by [19,75].
In brief, cells were seeded in 96-well plates (100 µL/well at a density of 1 × 10 4 cells/mL) and incubated in 5% CO 2 at 37 • C for 24 h. Cells were treated in triplicate with various concentrations of the tested extract and fractions after 24 h. The viable cell yield was determined by a colorimetric method as follows: after additional 24 h, the supernatant was removed and a crystal violet solution (1%) was added to each well for at least 30 min. Then, the stain was removed and the plates were washed using tap water until all the excess stains were removed. Next, 30% Glacial acetic acid was added to the whole wells and mixed. The absorbance of the plates was measured after gently shaking the Microplate reader (TECAN, Inc., Morrisville, NC, USA), using a test wavelength of 490 nm. Compared with the untreated cells, the optical density was measured with the microplate reader (SunRise, TECAN, Inc, Morrisville, NC, USA) to determine the viable cell numbers. The following equation was used to determine the percentage of viability.
Cell viability % = [1 − (ODt/ODc)] × 100% (2) where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of the untreated cells. The relation between the surviving cells and drug concentration was plotted to obtain the survival curve of each tumor cell line after treatment with the specified extract or fraction. The concentration required to cause toxic effects in 50% of the intact cells (IC 50 ) was estimated from the graphic plots of the dose response curve for each concentration using the Graphpad Prism 5 software (Graphpad software, San Diego, CA, USA).

Antimicrobial Activity
The Cliona sp. extract and its subfractions' (CH, CD, and CE) antibacterial activities were investigated by using the well diffusion method, as described by [19,76], against Staphylococcus aureus (S. aureus, ATCC 5368) as Gram-positive bacteria and Escherichia coli (E. coli, ATCC 10536) and Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853) as Gramnegative bacteria. The activity was determined by measuring the inhibition zone diameter in mm from 3 independent experiments and the average was considered. Ciprofloxacin (100 µg/mL) was used as a standard antibacterial drug (Positive control). The antifungal activity was also studied against Candida albicans (C. albicans, ATCC 10231) as reported by [19]; Fluconazole (100 µg/mL) was used as a positive antifungal control. The tested samples were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 500 µg/mL. DMSO was used as a negative control. The Muller Hinton Agar (MHA) medium was used for bacterial strains and the PDA (Potato Dextrose Agar) medium was used for c. albicans. The wells were filled with 100 µL of the stock solution of each sample, and the standards and DMSO Cultures were incubated at 37 • C for 24-48 h for fungi and 14-18 h for bacteria [77,78]. The tested bacterial strains and candida albicans were obtained from the Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University, Egypt.

Minimum Inhibitory Concentration Determination (MIC)
The Cliona sp. extract and its subfractions' (CH, CD, and CE MIC) values were determined using the agar dilution method as explained by [19]. In summary, the CT extract and its CH, CD, and CE fractions were dissolved in DMSO (not more than 5% and 2.5% for bacteria and fungi, respectively) and serially diluted. A series of MHA plates (for the tested bacterial strains) and PDA plates (for C. albicans) containing different dilutions of each extract and fraction were prepared. The tested bacterial strains were grown overnight on MHA, and purified colonies were suspended in 0.9% saline. The bacterial inoculum turbidity was adjusted to 0.5 McFarland standard (2.5 × 10 8 cfu /mL) and then diluted to 1:10 with sterile saline. The prepared MHA plates were inoculated by delivering 2 µL of the prepared inoculum on their surfaces to obtain a final concentration of 10 4 cfu per spot [79]. C. albicans was streaked on PDA, and the purified colonies were suspended in saline. The turbidity was adjusted to 0.5 Mcfarland standard (5 × 10 6 cfu/mL) and was then diluted in a 1:10 dilution with saline. Prepared PDA containing different concentrations of CT extract and CH, CD, and CE fractions were inoculated by delivering 2 µL of the prepared inoculum; therefore, the final concentration of the inoculum was 10 3 per the produced spot. Inoculated plates were incubated at 30 • C for 24-48 h and were examined for the presence of microbial growth. MIC is the lowest concentration of the antimicrobial agent that inhibits growth completely [80,81].

Statistical Analysis
GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA) was used for plotting the collected data. One-way ANOVA followed by Dunnett's multiple comparisons test was used for the data analysis and statistical significance calculation. A p-value < 0.05 was considered statistically significant. The data show the mean ± SD of three biological replicas.

Conclusions
In the present study, the Cliona sp. total extract, and the dichloromethane and ethyl acetate fractions were investigated using the UPLC-ESI-MS/MS analysis, which revealed the tentative identification of 23, 16, and 24 compounds from the n-hexane (CH), dichloromethane (CD), and ethyl acetate (CE) fractions of Cliona sp., respectively. Isolation and structure confirmation revealed four major compounds (coprostanol, brassicasterol, stigmasterol, and taurine) from the previous fractions. The cytotoxic, antioxidant, and antimicrobial activities of the ethanolic total extract and its subfractions were also determined in vitro. Remarkably, ethyl acetate was the most potent cytotoxic fraction, while dichloromethane showed a broad antimicrobial activity against all of the tested strains. Notably, the Cliona sp. total ethanolic extract is very active against the Gram-positive bacteria S. aureus.
In conclusion, this is the first study that investigates the chemical composition and biological activities of Cliona sp. from the Red Sea, Egypt. Based on the previous results, the Cliona sp. extract and its fractions could be a promising source of potent cytotoxic, antioxidant, and antimicrobial natural agents for multidrug-resistant bacterial and fungal strains, as well as different cancers. Further studies are planned for the most potent fractions of this sponge to test their effects against different cancer cell lines and resistant microbial strains. Further studies are also planned to isolate and identify the bioactive compounds from each major fraction using advanced techniques such as preparative or semipreparative HPLC.